The effects of optical genetic techniques on new neurons through the Wnt/β-Catenin pathway / 中国应用生理学杂志

OBJECTIVE@#To investigate the effects of optical genetic techniques on new neurons through the Wnt/β-Catenin pathway.@*METHODS@#Neural stem cells (ESCs)were extracted from the cerebral cortex of fetal rat and transfected by lentivirus carrying DCX-ChR2-EGFP gene and the expression of DCX of newborn neurons differentiated from neural stem cells were observed. All cells were divided into 3 groups(n=9): control group, NSCs+EGFP and NSCs+ChR2 groups. The control group was normal cultured NSCs (NSCs group); the neural stem cells in NSCs+EGFP group were transfected with lentivirus carrying EGFP gene. The neural stem cells in NSCs+ChR2 group were infected with lentivirus carrying DCX-ChR2-EGFP gene. After 48 hours of lentivirus infection, 470 nm blue laser irradiation was performed for 3 consecutive days. NeuN positive cell density(the maturation of neural stem cells)and the ratio of NeuN/Hoechst in each group were observed. Western blot was used to detect the expression levels of MAP2, NeuN, Neurog2, NeuroD1 and GluR2. Western blot was used to detect the expressions of β-catenin and TCF4 associated with Wnt/β-catenin signaling channel. Verapamil (100 μmol/L, L-type calcium channel blockers) and Dkk1 (50 μg/ml, β-catenin inhibitor) were used to treat stem cells of the NSCs+ChR2 group and then the expressions of MAP2, NeuN, Neurog2, NeuroD1 and GluR were detected by Western blot.@*RESULTS@#After 3 days of 470 nm blue laser irradiation, NeuN positive cell density(the maturation of neural stem cells)and the ratio of NeuN/Hoechst, the expression levels of the protein MAP2, NeuN, Neurog2, NeuroD1, GluR and the protein β-catenin and TCF4 associated with Wnt/β-catenin signaling channel detected by Western blot were significantly increased in the group of NSCs+ChR2, compared with NSCs and NSCs+EGFP groups. The expressions of MAP2, NeuN, Neurog2, NeuroD1 and GluR were remarkably decreased after treated by verapamil and Dkk1 in the group of NSCs+ChR2. It was proved that the opening of ChR2 channel producing cationic influx promoted the maturation of neural stem cells and induced by the Wnt/β-catenin signaling pathway.@*CONCLUSION@#Optical genetic promoted the maturation of newborn neurons through the Wnt/β-catenin signaling pathway.

Comparative study on sepsis models induced by Escherichia coli subtypes / 天津医药

Objective To investigate the degrees of injury severity of sepsis models made by different kinds of Escherichia coli. Methods The 152 mice were randomly divided into control group, DH5α group, 44102 group, and 25922 group, with 38 rats in each group. DH5α group, 44102 group and 25922 group were intraperitoneally injected with 300 μL of Escherichia coli DH5α, 44102 and 25922 at the concentration of 1.0 × 109CFU/kg to prepare sepsis models of different kinds of Escherichia coli. Control group was injected intraperitoneally with the same amount of normal saline. (1) After 8 h, four mice were taken from each group for peripheral blood bacterial culture . (2) After 12 h, ten mice in each group were used for measuring serum levels of TNF-α and IL-6 by enzyme-linked immunosorbent assay (ELISA). (3) Western blot assay was used to determine the serum levels of high-mobility group protein (HMGB1) in four mice of each group. (4) Ten mice in each group were used to measure serum levels of alanine transaminase (ALT), aspartate aminotransferase (AST), creatinine (CR) and blood urea nitrogen (BUN) by automatic biochemical analyzer. (5) After liver, lung and kidney tissues were fixed with formaldehyde, hematoxylin-eosin (HE) staining was performed (n=10 for each group). Results In DH5α group, 44102 group and 25922 group, bacteria, inflammatory cytokines TNF-α, IL-6 and HMGB1 protein, liver and kidney indicators ALT, AST, CR and BUN showed a sequential increasing trend (P＜0.01). The severe degrees of alveolar structure damage, hepatic cell infiltration and renal glomerular atrophy were DH5α group, 44102 group and 25922 group in turn. There were no obvious damages of lung, liver or kidney tissues in control group. Conclusion Escherichia coli 25922 induces severe sepsis injury and can be used to study the animal models of the initial inflammatory phase of sepsis. Escherichia coli 44102 induces moderate damage of sepsis and can be used in animal models that do not require definitive sepsis staging experiments. Escherichia coli DH5α induces less damage of sepsis and can be used to explore immunosuppressive therapy of the animal model of sepsis.